As inferred from the homologous UBA1, activation of ubiquitin by UBA6 (UBE1L1) proceeds through 3 steps: adenylation of ubiquitin, conjugation of ubiquitin from adenyl-ubiquitin to an internal cysteine residue of UBA6, and adenylation of a second molecule of ubiquitin. In the third step, the UBA6-ubiquitin conjugate adenylates the C-terminal glycine of a second ubiquitin molecule. This results in UBA6 loaded with 2 ubiquitin molecules, one of which is conjugated to an internal cysteine residue of UBA6 and one of which is adenylated and non-covalently bound to UBA6 (Jin et al. 2007, Pelzer et al. 2007, reviewed in Groettrup et al. 2008).